PROJECT SUMMARY Inter-tumoral heterogeneity ? the fact that every tumor has distinct genetic, epigenetic, and stromal features ? poses a challenge for precision cancer therapy. While genetic variants that influence a tumor?s response to targeted therapy (i.e. ?actionable mutations?) have received great attention, comparatively less is known about the causes of variation in the tumor microenvironment (TME). Nevertheless, such variation in the TME is likely to be of critical importance, as prior work has shown that the response to immunotherapy correlates with the abundance of T cells and other immune populations within a tumor [1,2]. We and others have shown, in the context of pancreatic ductal adenocarcinoma (PDA), that tumor-derived factors shape the microenvironment in vivo, dictating the relative abundance of different stromal populations. For example, tumor-derived GM-CSF recruits myeloid cells to the tumor, fostering an immunosuppressive environment [3,4], while tumor-derived Sonic Hedgehog causes an accumulation of myofibroblasts and other changes in the TME [5,6]. However, additional cancer cell-derived factors that act in similar fashion remain to be identified, representing an unexploited source of novel targets. In our preliminary work, we found that human PDA exhibits a wide spectrum of immune activity. Surprisingly, and in contrast to other tumor types, the presence or absence of an active immune signature was unrelated to the neo-antigen burden of a given tumor. We therefore hypothesize that cancer cell-intrinsic factors critically shape the immune microenvironment and drive immune heterogeneity. We further hypothesize that (i) a tumor?s immune makeup determines its response to immunotherapy and (ii) anti-tumor responses can be improved by modulating the immune infiltrate. Here, we propose an innovative approach to delineate the biology of immune heterogeneity in PDA, including novel implantable and genetically engineered mouse models studied in parallel with samples from an extensive tumor bank and prospectively collected from two clinical immunotherapy trials. Cellular, molecular, and clinical consequences of inter-tumoral heterogeneity will be assessed in the context of response to immunotherapy. Our ultimate goal is to understand and manipulate the immune microenvironment in PDA for therapeutic benefit, and we will approach this goal through the following three interrelated Specific Aims: Aim 1. Identify the molecular mechanism(s) underlying heterogeneity of immune infiltration Aim 2. Assess the impact of immune heterogeneity on the response to immunotherapy Aim 3. Elucidate the causes and consequences of immune heterogeneity in human PDA